PHYSIOLOGICAL ADAPTATION TO NUTRIENT LIMITATION IN A MARINE OLIGOTROPHIC ULTRAMICROBACTERIUM Sphingopyxis alaskensis MARTIN OSTROWSKI A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences Faculty of Science The University of New South Wales, Australia October 2006 UNIVERSITY OF NEW SOUTH WALES Thesis/Project Report Sheet Surname or Family name: OSTROWSKI First name: MARTIN Other name/s: - LUKE Abbreviation for degree as given in the University calendar: PhD School:BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES Faculty: SCIENCE Title: PHYSIOLOGICAL ADAPTATION TO NUTRIENT LIMITATION IN A MARINE OLIGOTROPHIC ULTRAMICROBACTERIUM Sphingopyxis alaskensis Abstract 350 words maximum: Sphingopyxis (formerly Sphingomonas) alaskensis, a numerically abundant species isolated from Alaskan waters and the North Sea represents one of the only pure cultures of a typical oligotrophic ultramicrobacterium isolated from the marine environment. In this study, physiological and molecular characterization of an extinction dilution isolate from the North Pacific indicate that it is a strain of Sphingopyxis alaskenis, extending the known geographical distribution of this strain and affirming its importance as a model marine oligotroph. Given the importance of open ocean systems in climatic processes, it is clearly important to understand the physiology and underlying molecular biology of abundant species, such as S. alaskensis, and to define their role in biogeochemical processes. S. alaskensis is thought to proliferate by growing slowly on limited concentrations of substrates thereby avoiding outright starvation. In order to mimic environmental conditions chemostat culture was used to study the physiology of this model oligotroph in response to slow growth and nutrient limitation. It was found that the extent of nutrient limitation and starvation has fundamentally different consequences for the physiology of oligotrophic ultramicrobacteria compared with well-studied copiotrophic bacteria (Vibrio angustum S14 and Escherichia coli). For example, growth rate played a critical role in hydrogen peroxide resistance of S. alaskensis with slowly growing cells being 10, 000 times more resistant than fast growing cells. In contrast, the responses of V. angustum and E. coli to nutrient availability differed in that starved cells were more resistant than growing cells, regardless of growth rate. In order to examine molecular basis of the response to general nutrient limitation, starvation and oxidative stress in S. alaskensis we used proteomics to define differences in protein profiles of chemostat-grown cultures at various levels of nutrient limitation. High-resolution two-dimensional electrophoresis (2DE) methods were developed and 2DE protein maps were used to define proteins regulated by the level of nutrient limitation. A number of these proteins were identified with the aid of mass spectrometry and cross-species database matching. The identified proteins are involved in fundamental cellular processes including protein synthesis, protein folding, energy generation and electron transport, providing an important step in discovering the molecular basis of oligotrophy in this model organism. Declaration relating to disposition of project report/thesis I am fully aware of the policy of the University relating to the retention and use of higher degree project reports and theses, namely that the University retains the copies submitted for examination and is free to allow them to be consulted or borrowed. Subject to the provisions of the Copyright Act 1968, the University may issue a project report or thesis in whole or in part, in photostat or microfilm or other copying medium. I also authorise the publication by University Microfilms of a 350 word abstract in Dissertation Abstracts International (applicable to doctorates only). .................................................... .................................................... .................................................... Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing to the Registrar. Requests for a longer period of restriction may be considered in exceptional circumstances if accompanied by a letter of support from the Supervisor or Head of School. Such requests must be submitted with the thesis/project report. FOR OFFICE USE ONLY Date of completion of requirements for Award Registrar and Deputy Principal Copyright and DAI Statement ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral thesis only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signature: ________________________ Date: __________________ Authenticity Statement ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signature: ________________________ Date: __________________ Table of Contents I List of Figures V List of Tables VII List of Abbreviations VIII Certificate of Originality X Acknowledgements XI Publications arising from this work XII ABSTRACT XIV 1 GENERAL INTRODUCTION ........................................................................1 1.1 INTRODUCTION ........................................................................................................... 2 1.2 OLIGOTROPHIC ENVIRONMENTS .................................................................................. 3 1.2.1 General observations.............................................................................3 1.2.1 An overview of the marine microbial community ...................................4 1.2.2 Factors affecting microbial growth........................................................8 1.3 DISTINCTIONS AND DEFINITIONS ................................................................................12 1.3.1 The size of small organisms.................................................................13 1.3.2 Copiotrophs vs Oligotrophs.................................................................14 1.4 OLIGOTROPHIC ISOLATES ...........................................................................................16 1.4.1 Predicted properties of oligotrophs .....................................................16 1.4.2 The extinction dilution method for isolating oligotrophs ......................17 1.5 DESCRIPTION OF SPHINGOMONAS ALASKENSIS RB2256..............................................21 1.5.1 Isolation of S. alaskensis......................................................................21 1.5.2 General growth characteristics ...........................................................23 1.5.3 Starvation and stress resistance...........................................................25 1.6 HYPOTHESIS ..............................................................................................................26 1.6.1 Overall objectives................................................................................27 1.6.2 Specific aims .......................................................................................27 2 MOLECULAR AND CHEMOTAXONOMIC CHARACTERISATION OF AN ABUNDANT ULTRAMICROBACTERIUM ISOLATED FROM THE OLIGOTROPHIC NORTH PACIFIC................................................................29 2.1 BACKGROUND ...........................................................................................................30 2.2 MATERIALS AND METHODS........................................................................................32 2.2.1 Bacterial strains and culture conditions ..............................................32 2.2.2 Alcohol precipitation...........................................................................32 2.2.3 Estimation of nucleic acid concentration .............................................32 2.2.4 Restriction enzyme digestion................................................................33 2.2.5 Agarose gel electrophoresis and visualisation .....................................33 2.2.6 Genomic DNA extraction.....................................................................34 2.2.7 Amplification of 16S rDNA sequences .................................................34 2.2.8 Purification and nucleotide sequencing of amplification products .......35 2.2.9 Phylogenetic analysis of 16S rDNA gene sequences.............................35 2.2.10 DNA-DNA hybridisation and mol%G+C content analysis .................35 2.2.11 Fatty acid analysis.............................................................................36